Project Summary Multiple myeloma (MM) is a deadly disease characterized by a shortage of red blood cells, white blood cells, and platelets in the blood.1,2 The disease has a grim prognosis, as it is estimated that in 2018 there were 30,770 new cases and 12,770 deaths resulting from MM.3 In the past decade there have been significant strides made in the development of new drugs and implementation of combination therapies, but variability in treatment response between patients has kept median survival approximately 5 years and MM remains an incurable disease.1,2,4 MM has not only been shown to be a highly heterogenous disease from patient-to- patient, but patients have also exhibited intra-tumor clonal heterogeneity.4?8 For this reason, personalized therapies are highly desirable, particularly those targeted at specific signaling pathways exhibiting aberrant behavior in a given patient.1,2,9,10 Presently, cytogenetic and molecular markers are used to inform personalized therapies; however, these assays do not directly investigate specific biochemical activity i.e. the target for most pharmacologic interventions. It is well-accepted that the enzyme sphingosine kinase (SK) plays a crucial function in MM initiation, progression, and drug resistance.11?20 This knowledge, in combination with the increasing number of single-cell studies in MM highlighting that the heterogeneity of the malignancy from patient-to-patient and intra-tumor clonal heterogeneity affects MM cell sensitivity to various drugs, suggests that measurements of metabolic activity in the sphingolipid pathway will improve MM treatment response and decrease the frequency of relapse.4?7,21,22 Biochemical investigations of the sphingolipid pathway have traditionally relied on bulk cell assays, which only reflect a population average of cell behavior.23?26 Single-cell assays have been developed, but even highly automated assays have been low throughput and technically complex.27?29 I aim to address the limitations of existing SK assays by creating an increased-throughput, highly parallelizable, simple-to-use chemical separations platform that incorporates a chemical sensor or reporter for enzyme activity measurement. The analytical chemistry technology proposed in this research will facilitate measurements of SK activity within samples of primary MM cells, providing insights into the efficacy of SK inhibitors as part of a therapy regimen on a patient-by-patient basis. The central hypothesis of this research is that MM cells analyzed on the proposed novel single-cell analysis platform will exhibit heterogeneity in SK activity and diversity in responding to SK inhibitors. It is important to note that while this proposal limits its scope to investigating SK activity in MM cells, the platform can easily be adapted to investigate essentially any metabolic pathway for any disease, provided that chemical probes are available. This urgently needed novel technology will provide researchers with an easy-to-use, increased throughput assay for evaluating biochemical activity within single cells.